The invention relates to a new polynucleotide, the polypeptide encoded by said polynucleotide, the uses of said polynucleotide and polypeptide, and the methods for preparing same. In particular, the polypeptide of the invention is identified as a new member of the lysozyme family.
Lysozyme exists ubiquitously in all parts of organisms, including various tissues, organs, and sera; it is especially abundant in egg white. Lysozyme is mainly secreted by the epithelial cell of certain glands and some kinds of leukocyte.
Lysozyme was first reported by Fleming, et al. in 1922. Afterward, lysozyme has been widely studied. A lot of papers concerning its crystal structure, protein catalytic domains, catalytic dynamics, immunology, molecular evolutionary, and so on, have been published. Lysozyme is one of the proteins that are studied most extensively and intensively. However, the study on lysozyme gene is not yet sufficient. Nowadays, only a few lysozyme genes from different species, such as E.coli T4, salmonella P22 phage, bacillus xcfx86 phage and chicken, etc., have been cloned. (1983 J. Mol. Biol. 165. 229-248; 1985 Virology 143, 280-289; 1987 Proc. Natl. Acad. Sci. USA, 77, 5759-5763). The cloning about human lysozyme gene was also reported (1988, Gene 66,223-234).
The main function of lysozyme is to hydrolyze the beta(1-4) glycosidic bond between N-acetylmuramic acid (NAM) and N-acetylgluconic acid (NAG) of the bacterial cell wall. In the organism, lysozyme can act as a nonspecific immune molecule against bacterial infections, and as a digestive enzyme in enteron and some mollusks which live on bacteria. Further, lysozyme has the function of inhibiting tumor growth. Therefore, lysozyme has important applications in both industry and medicine.
One purpose of the invention is to provide a new polynucleotide which encodes a new member of lysozyme gene family. The new human lysozyme is named LYC3.
Another purpose of the invention is to provide a new member of lysozyme protein family, which is named LYC3.
Still another purpose of the invention is to provide a new method for preparing said new human lysozyme by recombinant techniques.
The invention also relates to the uses of said human lysozyme and its coding sequence.
In one aspect, the invention provides an isolated DNA molecule, which comprises a nucleotide sequence encoding a polypeptide having human LYC3 protein activity, wherein said nucleotide sequence shares at least 70% homology to the nucleotide sequence of nucleotides 81-521 in SEQ ID NO: 3, or said nucleotide sequence can hybridize to the nucleotide sequence of nucleotides 81-521 in SEQ ID NO: 3 under moderate stringency. Preferably, said nucleotide sequence encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 4 or of amino acids 19-146 of SEQ ID NO: 4. More preferably, the sequence comprises the nucleotide sequence of nucleotides 81-521 in SEQ ID NO: 3.
Further, the invention provides an isolated LYC3 polypeptide, which comprises a polypeptide having the amino acid sequence of SEQ ID NO: 4 or of amino acids 19-146 of SEQ ID NO: 4, its active fragments, and its active derivatives. Preferably, the polypeptide is a polypeptide having the amino acid sequence of SEQ ID NO: 4.
The invention also provides a vector comprising said isolated DNA.
The invention further provides a host cell transformed with said vector.
In another aspect, the invention provides a method for producing a polypeptide with the activity of LYC3 protein, which comprises:
(a) forming an expression vector of LYC3 protein comprising the nucleotide sequence encoding the polypeptide having the activity of LYC3 protein, wherein said nucleotide sequence is operably linked with an expression regulatory sequences, and said nucleotide sequence shares at least 70% homology to the nucleotide sequence of positions 81-521 in SEQ ID NO: 3;
(b) introducing the vector of step (a) into a host cell, thereby forming a recombinant cell of LYC3 protein;
(c) culturing the recombinant cell of step (b) under the conditions suitable for the expression of LYC3 polypeptides;
(d) isolating the polypeptides having the activity of LYC3 protein.
In one embodiment of the present invention, the isolated polynucleotide has a full length of 544 nucleotides, whose detailed sequence is shown in SEQ ID NO: 3. The open reading frame (ORF) locates at nucleotides 81-521.
In the present invention, the term xe2x80x9cisolatedxe2x80x9d or xe2x80x9cpurifiedxe2x80x9d or xe2x80x9csubstantially purexe2x80x9d DNA refers to a DNA or fragment which has been isolated from the sequences which frank it in a naturally occurring state. The term also applied to DNA or DNA fragment which has been isolated from other components naturally accompanying the nucleic acid and from proteins naturally accompanying it in the cell.
In the present invention, the term xe2x80x9cLYC3 protein encoding sequencexe2x80x9d or xe2x80x9cLYC3 polypeptide encoding sequencexe2x80x9d refers to a nucleotide sequence encoding a polypeptide having the activity of LYC3 protein, such as the nucleotide sequence of positions 81-521 in SEQ ID NO: 3 or its degenerate sequence. The degenerate sequences refer to the sequences formed by replacing one or more codons in the ORF of 81-521 in SEQ ID NO: 3 with degenerate codes which encode the same amino acid. Because of the degeneracy of codon, the sequence having a homology as low as about 70% to the sequence of nucleotides 81-521 in SEQ ID NO: 3 can also encode the sequence shown in SEQ ID NO: 4. The term also refers to the nucleotide sequences that hybridize with the nucleotide sequence of nucleotides 81-521 in SEQ ID NO: 3 under moderate stringency or preferably under high stringency. In addition, the term also refers to the sequences having a homology at least 70%, preferably 80%, more preferably 90% to the nucleotide sequence of nucleotides 81-521 in SEQ ID NO: 3. Moreover, the term includes a nucleotide sequence encoding a mature protein without signal peptide, such as the nucleotide sequence of position 135-521 in SEQ ID NO: 3.
The term also refers to variants of the sequence in SEQ ID NO: 3, which are capable of coding for a protein having the same function as human LYC3 protein. These variants includes, but are not limited to: deletions, insertions and/or substitutions of several nucleotides (typically 1-90, preferably 1-60, more preferably 1-20, and most preferably 1-10) and additions of several nucleotides (typically less than 60, preferably 30, more preferably 10, most preferably 5) at 5xe2x80x2 end and/or 3xe2x80x2 end.
In the present invention, xe2x80x9csubstantially purexe2x80x9d proteins or polypeptides refers to those which occupy at least 20%, preferably at least 50%, more preferably at least 80%, most preferably at least 90% of the total sample material (by wet weight or dry weight). Purity can be measured by any appropriate method, e.g., in the case of polypeptides by column chromatography, PAGE or HPLC analysis. A substantially purified polypeptides is essentially free of naturally associated components.
In the present invention, the term xe2x80x9cLYC3 polypeptidexe2x80x9d or xe2x80x9cLYC3 proteinxe2x80x9d refers to a polypeptide having the activity of LYC3 protein comprising the amino acid sequence of SEQ ID NO: 4 or of amino acids 19-146 of SEQ ID NO: 4. The term also comprises the variants of said amino acid sequence which have the same function of human lysozyme. These variants include, but are not limited to, deletions, insertions and/or substitutions of several amino acids (typically 1-50, preferably 1-30, more preferably 1-20, most preferably 1-10), and addition of one or more amino acids (typically less than 20, preferably less than 10, more preferably less than 5) at C-terminal and/or N-terminal. For example, the protein function are usually unchanged when an amino residue is substituted by a similar or analogous one. Further, the addition of one or several amino acids at C-terminal and/or N-terminal will not change the function of protein. The term also includes the active fragments and derivatives of LYC3 protein.
The variants of polypeptide include homologous sequences, allelic variants, natural mutants, induced mutants, proteins encoded by DNA which hybridizes to LYC3 DNA under high or low stringency conditions as well as the polypeptides or proteins retrieved by antisera raised against LYC3 polypeptide. The present invention also provides other polypeptides, e.g., fusion proteins, which include the LYC3 polypeptide or fragments thereof. In addition to substantially full-length polypeptide, the soluble fragments of LYC3 polypeptide are also provided. Generally, these fragments comprise at least 10, typically at least 30, preferably at least 50, more preferably at least 80, most preferably at least 100 consecutive amino acids of human LYC3 polypeptide.
The present invention also provides the analogues of LYC3 protein or polypeptide. Analogues can differ from naturally occurring LYC3 polypeptide by amino acid sequence differences or by modifications which do not affect the sequence, or by both. These polypeptides include genetic variants, both natural and induced. Induced variants can be made by various techniques, e.g., by random mutagenesis using irradiation or exposure to mutagens, or by site-directed mutagenesis or other known molecular biologic techniques. Also included are analogues which include residues other than those naturally occurring L-amino acids (e.g., D-amino acids) or non-naturally occurring or synthetic amino acids (e.g., beta- or gamma-amino acids). It is understood that the polypeptides of the invention are not limited to the representative polypeptides listed hereinabove.
Modifications (which do not normally alter primary sequence) include in vivo, or in vitro chemical derivation of polypeptides, e.g., acelylation, or carboxylation. Also included are modifications of glycosylation, e.g., those made by modifying the glycosylation patterns of a polypeptide during its synthesis and processing or in the further processing steps, e.g., by exposing the polypeptide to enzymes which affect glycosylation (e.g., mammalian glycosylating or deglycosylating enzymes). Also included are sequences which have phosphorylated amino acid residues, e.g., phosphotyrosine, phosphoserine, phosphothronine, as well as sequences which have been modified to improve their resistance to proteolytic degradation or to optimize solubility properties.
The invention also includes antisense sequence of the sequence encoding LYC3 polypeptide. Said antisense sequence can be used to inhibit expression of LYC3 in cells.
The invention also include probes, typically having 8-100, preferably 15-50 consecutive nucleotides. These probes can be used to detect the presence of nucleic acid molecules coding for LYC3 in samples.
The present invention also includes methods for detecting LYC3 nucleotide sequences, which comprises hybridizing said probes to samples, and detecting the binding of the probes. Preferably, the samples are products of PCR amplification. The primers in PCR amplification correspond to coding sequence of LYC3 polypeptide and are located at both ends or in the middle of the coding sequence. In general, the length of the primers is 20 to 50 nucleotides.
A variety of vectors known in the art, such as those commercially available, are useful in the invention.
In the invention, the term xe2x80x9chost cellsxe2x80x9d includes prokaryotic and eukaryotic cells. The common prokaryotic host cells include Escherichi coli, Bacillus subtilis, and so on. The common eukaryotic host cells include yeast cells, insect cells, and mammalian cells. Preferably, the host cells are eukaryotic cells, e.g., CHO cells, COS cells, and the like.
In another aspect, the invention also includes antibodies, preferably monoclonal antibodies, which are specific for polypeptides encoded by LYC3 DNA or fragments thereof. By xe2x80x9cspecificityxe2x80x9d is meant an antibody which binds to the LYC3 gene products or a fragments thereof. Preferably, the antibody binds to the LYC3 gene products or a fragments thereof and does not substantially recognize and bind to other antigenically unrelated molecules. Antibodies which bind to LYC3 and block LYC3 protein and those which do not affect the LYC3 function are included in the invention. The invention also includes antibodies which bind to the LYC3 gene product in its unmodified as well as modified form.
The present invention includes not only intact monoclonal or polyclonal antibodies, but also immunologically-active antibody fragments, e.g., a Fabxe2x80x2 or (Fab)2 fragment, an antibody light chain, an antibody heavy chain, a genetically engineered single chain Fv molecule (Lander, et al., U.S. Pat. No. 4,946,778), or a chimeric antibody, e.g., an antibody which contains the binding specificity of a murine antibody, but the remaining portion of which is of human origin.
The antibodies in the present invention can be prepared by various techniques known to those skilled in the art. For example, purified LYC3 gene products, or its antigenic fragments can be administrated to animals to induce the production of polyclonal antibodies. Similarly, cells expressing LYC3 or its antigenic fragments can be used to immunize animals to produce antibodies. Antibodies of the invention can be monoclonal antibodies which can be prepared by using hybridoma technique (See Kohler, et al., Nature, 256; 495,1975; Kohler, et al., Eur. J. Immunol. 6: 511,1976; Kohler, et al., Eur. J. Immunol. 6: 292, 1976; Hammerling, et al., In Monoclonal Antibodies and T Cell Hybridomas, Elsevier, N.Y., 1981). Antibodies of the invention comprise those which block LYC3 function and those which do not affect LYC3 function. Antibodies in the invention can be produced by routine immunology techniques and using fragments or functional regions of LYC3 gene product. These fragments and functional regions can be prepared by recombinant methods or synthesized by a polypeptide synthesizer. Antibodies binding to unmodified LYC3 gene product can be produced by immunizing animals with gene products produced by prokaryotic cells (e.g., E. coli); antibodies binding to post-translationally modified forms thereof can be acquired by immunizing animals with gene products produced by eukaryotic cells (e.g., yeast or insect cells).
The full length human LYC3 nucleotide sequence or its fragment of the invention can be prepared by PCR amplification, recombinant method and synthetic method. For PCR amplification, one can obtain said sequences by designing primers based on the nucleotide sequence disclosed in the invention, especially the sequence of ORF, and using cDNA library commercially available or prepared by routine techniques known in the art as a template. When the sequence is long, it is usually necessary to perform two or more PCR amplifications and link the amplified fragments together in the correct order.
Once the sequence is obtained, a great amount of the sequences can be produced by recombinant methods. Usually, said sequence is cloned in a vector which is transformed into a host cell. Then the sequence is isolated from the amplified host cells using conventional techniques.
In addition to recombinant techniques, the protein fragments of the invention may also be prepared by direct chemical synthesis using solid phase synthesis techniques (Stewart et al., (1969) Solid-Phase Peptide Synthesis, WH Freeman Co., San Francisco; Merrifield J. (1963), J. Am. Chem. Assoc. 85: 2149-2154). In vitro protein synthesis can be performed manually or automatically, e.g., using a Model 431 Peptide Synthesizer (Applied Biosystems, Foster City, Calif.). The fragments of protein of the invention can be synthesized separately and linked together using chemical methods so as to produce full-length molecule.
The sequences encoding the protein of the present invention are also valuable for gene mapping. For example, the accurate chromosome mapping can be performed by hybridizing cDNA clones to a chromosome in metaphase. This technique can use cDNA as short as about 500 bp, or as long as about 2000 bp, or more. For details, see Verma et al., Human Chromosomes: A Manual of Basic Techniques, Pergamon Press, New York (1988).
Once a sequence has been mapped to a precise chromosomal location, the physical position of the sequence on the chromosome can be correlated with genetic map data. Such data are found in, e.g., Mendelian Inheritance in Man (available on-line through Johns Hopkins University Welch Medical Library). The relationships between genes and diseases that have been mapped to the same chromosomal region are then identified through linkage analysis.
Then, the differences in the cDNA or genomic sequence between affected and unaffected individuals can also be determined. If a mutation is observed in some or all of the affected individuals but not in any normal individual, then the mutation is likely to be the causative agent of the disease.
The substances which act with the LYC3, e.g., receptors, inhibitors and antagonists, can be screened out by various conventional techniques and using the protein of the invention.
The protein, antibody, inhibitor, antagonist or receptor of the invention provide different effects when administrated in therapy. Usually, these substances are formulated with a non-toxic, inert and pharmaceutically acceptable aqueous carrier. The pH typically ranges from 5 to 8, preferably about 6-8, although pH may alter according to the property of the formulated substances and the diseases to be treated. The formulated pharmaceutical composition is administrated in conventional routine including, but not be limited to, intramuscular, intraperitoneal, subcutaneous, intracutaneous, or topical administration.
As an example, the human LYC3 protein of the invention may be administrated together with the suitable and pharmaceutically acceptable carrier. The examples of carriers include, but are not limited to, saline, buffer solution, glucose, water, glycerin, or the combination thereof. The pharmaceutical formulation should be suitable for the delivery method. The human LYC3 protein of the invention may be in the form of injections which are made by conventional methods and using physiological saline or other aqueous solution containing glucose or auxiliary substances. The pharmaceutical compositions in the form of tablet or capsule may be prepared by routine methods. The pharmaceutical compositions, e.g., injections, solutions, tablets, and capsules, should be manufactured under sterile conditions. The active ingredient is administrated in therapeutically effective amount, e.g., from about 1 ug to 5 mg per kg body weight per day. Moreover, the polypeptide of the invention can be administrated together with other therapeutic agent.
When the human LYC3 polypeptides of the invention are used as a pharmaceutical, the therapeutically effective amount of the polypeptides are administrated to mammals. Typically, the therapeutically effective amount is at least about 10 ug/kg body weight and less than about 8 mg/kg body weight in most cases, and preferably about 10 ug-1 mg/kg body weight. Of course, the precise amount will depend upon the factors, such as delivery methods, the subject health, and the like, and is within the judgment of the skilled clinician.
In one embodiment, the polynucleotide of the invention is 544 bp in full length whose detailed sequence is shown in SEQ ID NO: 3 with the ORF located at positions 81-521. Said polynucleotide was obtained as follows: human brain gt 11 cDNA library (Clontech) was used as a template and PCR was carried out with the synthetic forward primer A1 5xe2x80x2-AGAGTGGTGGTGGCTCCACTCTG-3xe2x80x2 (SEQ ID NO. 1) and reverse primer B : 5xe2x80x2-TGCTGTGCATGGTTCCGTCCATC-3xe2x80x2 (SEQ ID NO. 2). A target fragment of 544 bp was obtained. The sequencing of the PCR product gave the full length cDNA sequence shown in SEQ ID NO:3.
Homology comparison showed that the nucleotide sequence and the coded protein sequence of the invention shared remarkable homology to other lysozymes from different origins. Therefore, it indicates it is a new member of lysozyme family and has some important functions of the family.
Lysozyme can lyse cells by hydrolyze the beta(1-4) glycosidic bond between N-acetylmuramic acid (NAM) and N-acetylgluconic acid (NAG) of the bacterial cell wall. In the organisms, lysozyme can act as a nonspecific immune molecule against bacterial infections, and as a digestive enzyme in enteron and some mollusks which live on bacteria. Further, lysozyme has the function of inhibiting tumor growth. In 1955, Caselli and Shumacher (Boll Ocul 34:513-533, 1955) reported on the lysozyme-mediated 70% inhibition of neoplastic transformation in cornea of chicken infected by Rous sarcoma virus. Many other experiments indicated that lysozyme participates in the process of tumor diffusion and interacts with phospho- and glucolipid molecule of tumor cells. The inhibition effect on human tumor of lysozyme was reported and patented (1980 Jpn Kokai, Tokkyo Koho 33,409; 1980 Jpn Kokai Tokkyo Koho 33,408). As to the mechanism of lysozyme inhibition on tumor, there are two possibilities: (1) lysozyme directly activates the organism""s immunity functions; (2) lysozyme indirectly enhances the organism""s immune ability (1989 Anticancer Research 9, 583-592).